Fractionation of macro-molecules using asymmetric pulsed field electrophoresis

ABSTRACT

A method and apparatus for fractionation of charged macro-molecules such as DNA is provided. DNA solution is loaded into a matrix including an array of obstacles. An alternating electric field having two different fields at different orientations is applied. The alternating electric field is asymmetric in that one field is stronger in duration or intensity than the other field, or is otherwise asymmetric. The DNA molecules are thereby fractionated according to site and are driven to a far side of the matrix where the fractionated DNA is recovered. The fractionating electric field can be used to load and recover the DNA to operate the process continuously.

RELATED APPLICATIONS

[0001] This application claims the priority of Provisional ApplicationSerial No. 60/256,298, filed Dec. 18, 2000, the entire disclosure ofwhich is expressly incorporated herein by reference.

GOVERNMENT RIGHTS

[0002] The present invention has been made under Federal Contract GrantNo. MDA 972-00-10031 and the government may have certain rights to thesubject invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of The Invention

[0004] The present invention relates to a method and apparatus forfractionating charged macro-molecules such as DNA using asymmetricpulsed field electrophoresis.

[0005] 2 . Related Arts

[0006] The analysis and fractionation of large DNA molecules is acentral step in large scale sequencing projects. Conventionally, gelelectrophoresis is used to fractionate DNA molecules according to theirsizes. This method includes two steps: sample loading and fractionation.First, sample solution containing DNA is loaded into loading wells inthe gel slab before the electric field is turned on. Then, an electricfield is applied. The DNA molecules move in the opposite direction ofthe electric field because they are negatively charged. As the electricfield is applied, DNA molecules travel at different speeds according totheir sizes, but the directions in which they migrate are always thesame. Eventually, sample DNA molecules are separated into differentbands, each of which contains DNA molecules of the same size, as shownin FIG. 1. Shorter DNA fragments move faster than longer ones.Therefore, they are separated according to their sizes. However, thisstandard method only works effectively for DNA molecules smaller than 40kbp. Above this range, the standard method has to be modified. Inparticular, the applied electric field can no longer be DC, but is madeto alternate between two different orientations. This modified scheme(pulsed-field gel electrophoresis) is routinely used in modern molecularbiology laboratories, but it typically takes a few days to fractionateone set of DNA samples.

[0007] What is needed, and has not heretofore been provided, is a methodand apparatus for quickly, or even continuously, fractionating chargedmacro-molecules.

OBJECTS AND SUMMARY OF THE INVENTION

[0008] It is an object of the present invention to provide a method andapparatus for quickly fractionating charged macro-molecules.

[0009] It is an additional object of the present invention to provide amethod and apparatus for continuously fractionating chargedmacro-molecules.

[0010] It is a further object of the present invention to provide amethod and apparatus for fractionating macro-molecules using asymmetricpulsed electrophoresis wherein an alternating electric field having twodifferent orientations is applied, and one of the fields is strongerthan the other in terms of duration or intensity, or the field isotherwise asymmetric.

[0011] The present invention relates to a method and apparatus forfractionation of charged macro-molecules such as DNA. DNA solution isloaded into a matrix including an array of obstacles. An alternatingelectric field having two different fields at different orientations isapplied. The alternating electric field is asymmetric in that one fieldis stronger in duration or intensity than the other field, or isotherwise asymmetric. The DNA molecules are thereby fractionatedaccording to size and are driven to a far side of the matrix where thefractionated DNA is recovered. The fractionating electric field can beused to load and recover the DNA to operate the process continuously.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Other important objects and features of the invention will beapparent from the following Detailed Description of the Invention takenin connection with the accompanying drawings in which:

[0013]FIG. 1 shows conventional gel electrophoresis.

[0014]FIG. 2 is a diagram showing asymmetric pulsed-fieldelectrophoresis in micro/nano-fabricated matrices according to thepresent invention.

[0015]FIG. 3 is a diagram showing the basic principle of asymmetricalpulsed electrophoresis of the present invention.

[0016]FIG. 4 shows the way stretched DNA molecules move underasymmetrical pulsed electric field.

[0017]FIG. 5 shows a support material (matrix) for use in fractionationof DNA according to the present invention.

[0018]FIG. 6A is a top view and FIG. 6B is a side view of themicrofabricated support material shown in FIG. 5.

[0019]FIG. 7 shows fractionation of T4 and T7 DNA.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention relates to a method and apparatus forfractionation of charged macro-molecules such as DNA. DNA solution isloaded into a matrix including an array of obstacles. An alternatingelectric field having two different fields at different orientations isapplied. The alternating electric field is asymmetric in that one fieldis stronger in duration or intensity than the other field, or isotherwise asymmetric. The DNA molecules are thereby fractionatedaccording to size and are driven to a far side of the matrix where thefractionated DNA is recovered. The fractionating electric field can beused to load and recover the DNA to operate the process continuously.

[0021] The present invention provides a method and apparatus for thefractionation of macromolecules on micro/nano-fabricated supportmaterials (a.k.a. matrices). Because the motion of DNA molecules can beaccurately controlled in micro/nano-fabricated environments, thefractionation of DNA can be achieved with very high resolution in ashort time (i.e. seconds), even for DNA molecules larger than 100 kbp.In addition, the process can be operated continuously, i.e., DNA isloaded, fractionated, and recovered at the same time. Moreover, becausethis method exploits micro/nano-fabricated structure, it can be readilyintegrated into lab-on-a-chip devices as a component.

[0022] According to the present invention, DNA molecules enter from onepoint or loading channel 14 on the boundary 12 of the matrix 10 as shownin FIG. 2. The molecules are subsequently fractionated into differentbands at different orientations, according to their sizes, as they aredriven towards the other side 13 of the matrix 10, where the purifiedDNA molecules 30 are finally recovered. The DNA molecules arefractionated into short fragments 32 at one end, long fragments 36 atthe other end, and medium fragments 34 therebetween. The electric field(E₁ and E₂) that fractionates the DNA sample can also be used to loadand recover the sample, enabling the process to be operatedcontinuously.

[0023] A mixture of DNA molecules emerges continuously from the loadingchannel. The support material comprises a micro/nano-fabricated porousstructure, in which DNA molecules can move. An alternating electricfield, shown in E₁ and E₂, is applied across the whole matrix. E₁ and E₂are at an angle with respect to each other, preferably an obtuse angle,and have different intensities and/or durations. Because DNA moleculesare stretched and moving in a zigzag way under the alternating field,shorter fragments move at an angle to longer fragments.

[0024] When DNA molecules are subject to an alternating electric fieldbetween two orientations at an angle such as an obtuse angle, they arestretched to different lengths according to their molecular weight.Referring to FIG. 3, let the end-to-end length of a stretched DNAmolecule be x. Assume that electric field E₁ displaces every DNAmolecules by approximately the same displacement α e₁, whereas E₂displaces every DNA molecules by approximately β e₂ (e₁ and e₂ are unitvectors, and both α and β are positive numbers, since DNA molecules arenegatively charged and move opposite to an applied electric field). Thisis a valid assumption because it is known that all DNA molecules havevirtually the same mobility due to the fact that the long rangehydrodynamic interaction is shielded by the counter ion layers. For thesimplicity, let β be larger than β. This can be achieved by pulsingalong −e₁ longer than along −e₂, and/or by making the electric fieldstronger along −e₁ than along −e₂. Because the electric field isalternating between two different directions, the DNA molecules willmove in a zigzag way. Ideally, the electric field is chosen so thatx<β<α. The net motion of very short DNA molecules (x<<β) in one pulsingcycle (a cycle refers to applying E₁, then E₂) is simply αe₁+β e₂. Onthe contrary, very long molecules (x>β) travel (α−β) e₁ in a cycle. Eventhough this could be rather surprising at first glance, it is not hardto understand if it is realized that when the field is switched from oneto the other, the tails 40 of DNA strands become the ends that lead themotion and the heads 42 follow, as shown in FIG. 4. In principle, we canpredict the angles of the bands into which DNA mixtures are fractionatedby this technique, if the stretched lengths of DNA molecules are smallerthan or equal to β. Within this range (x<β or x=β), the net motion ofDNA molecules in one cycle is (α−x) e₁+(β−x) e₂. Purified DNA moleculescan be recovered at the bottom of the support material, after manycycles. In one cycle, a DNA molecule stretched to length x will travel(α−x) e₁+(β−x) e₂.

[0025] As shown in FIG. 4, an alternating electric field not onlystretches DNA molecules to a linear conformation, but also makes them tomove in a zigzag way. The initial position of a DNA molecule is labeledas 0. The big dot on one end of the DNA represents the “head” 42 of themolecule. The other end of the molecule is referred to as the “tail” 40.When E₁ is applied, the DNA molecule moves to position 1. The tail 40leads the motion as the electric field is switched to E₂. By the end ofone cycle, the molecule moves to position 2, and the net displacement inone cycle (α−x) e₁+(β−x) e₂.

[0026] By electric field, what is meant is the spatial average of thefield around a location over a length scale of several obstacles, notthe microscopic field distribution around a single obstacle. Anyelectric field at a given location, whose direction varys with time, canbe resolved uniquely into two sequences of electric pulses according tothe instantaneous direction of the field. The first sequence of electricpulses comprises the electric field pointing to one side of the averagefield vector over the whole period of time when the field is applied tofractionate the molecules. The second sequence of electric pulsescomprises the electric field pointing to the other side of the averagefield vector. If the field vector at a moment is at the same directionor at the opposite direction of the average field vector, it is excludedin either of the pulse sequence. By asymmetrical electric field, what ismeant is that the two sequences of electric pulses, resolved from agiven electric field, as a function of time, have vector integrals overtime that is not symmetric about the time-averaged field direction. Saidanother way, the electric fields, fields {right arrow over (E)}(t) whoseodd-order integrals over time, ∫|E(t)|E(t)dt, are not at thetime-average field orientation for every n, where n is any positive eveninteger. As such, by applying electric fields with differentorientations and different strengths, i.e. different durations ordifferent intensities or both, one applies an asymmetric field.Asymmetric fields can also be generated by sweeping signals in terms oforientation, duration and intensity. In the past, the field has firstand second pulse sequences whose vector integrals over time aresymmetrical about the average field.

[0027] Experimental Results

[0028] The following example uses a microfabricated matrix 10. As shownin FIG. 5, the matrix 10 consists of two parts: a microfabricated arrayof obstacles 20 in quartz, and a cap layer 18 that is hermeticallybonded to the microfabricated side of the quartz substrate 16. Thequartz substrate 16 is surface-micromachined using standardmicrofabrication techniques. The substrate is subsequently bonded to aglass cap layer 18 hermetically. The cavities between the substrate andthe cap layer become microfluidic channels in which DNA molecules arefractionated. The dimensions of this microfabricated device are depictedin FIGS. 6a and 6 b. FIG. 6a is the top view of the matrix 10, and FIG.6b is a side view of the matrix 10. The matrix 10 in this case is ahexagonal array of obstacles 20. Each obstacle 20 comprises acylindrical post 2 μm in diameter. The center-to-center distance betweenneighboring obstacles is 4 μm. The uniformity of the electric fieldacross the whole matrix is controlled accurately by the peripheralstructures surrounding the matrix. FIG. 7 shows the fractionation of T4(169 kbp) and T7 (40 kbp) DNA molecules. The pulse condition is E₁=120V/cm at 60° with respect to the horizontal boundary, and E₂=60 V/cm at−60°. The DNA injected into the matrix is 10 μg/ml of T4 DNA and 10μg/ml of T7 DNA in ½ TBE buffer. The duration of E₁ is identical to thatof E₂, which is 166 msec. The frequency at which the electric fieldalternates is 3 Hz. Clearly, the DNA mixture separates into two bands.

[0029] Having thus described the invention in detail, it is to beunderstood that the foregoing description is not intended to limit thespirit and scope thereof. What is desired to be protected by LettersPatent is set forth in the appended claims.

What is claimed is:
 1. A method of fractionating charged macro-moleculescomprising: loading molecules into a matrix of obstacles; and applyingan electric field, which varies asymmetrically, to the matrix.
 2. Themethod of claim 1 wherein the step of applying an asymmetric electricfield to the matrix comprises applying an electric field which isalternating in direction as a function of time at a location in thematrix, and which has a time average of its vector over many cycles,whereby the time integral of the electric field vector at the samelocation over the part of the cycles when it is instantaneously pointingto one side of the said time-averaged electric field vector is notspatially symmetric about the same said time-averaged vector with thetime integral of the electric field vector over the part of the cyclesat the same location when it is instantaneously pointing to the otherside of the same said time-averaged vector.
 3. The method of claim 1wherein the step of applying an asymmetric electric field to the matrixcomprises applying to the matrix time-dependent electric fields {rightarrow over (E)}(t) whose odd-order integrals over time, ∫|{right arrowover (E)}(t)|^(n){right arrow over (E)}(t)dt, are not at thetime-average field orientation for every n, where n is any positive eveninteger.
 4. The method of claim 1 wherein the electric fields comprise:alternating first and second electric pulses of first and secondwaveforms; maintaining the integral of one of the first or secondpulses' amplitude over time larger than that of the other pulse; varyingthe orientation of the first electric pulse within first orientation andsecond orientation, and the orientation of the second electric pulsewithin third orientation and forth orientation.
 5. The method of claim 4wherein the first and second waveforms are square pulses.
 6. The methodof claim 5 wherein one of the square pulses is of higher amplitude thanthe other.
 7. The method of claim 5 wherein one of the square pulses isof longer duration than the other.
 8. The method of claim 1 wherein theelectric fields comprise: alternating first and second electric pulsesof first and second waveforms; maintaining the integral over time of oneof the first or second pulses' amplitudes larger than that of the otherpulse; applying the first and second electric pulses at first and secondfixed orientations.
 9. The method of claim 8 wherein the first andsecond waveforms are square pulses.
 10. The method of claim 9 whereinone of the square pulses is of higher amplitude than the other.
 11. Themethod of claim 9 wherein one of the square pulses is of longer durationthan the other.
 12. The method of claim 1 wherein the chargedmacro-molecules are deoxyribonucleic acid (a.k.a. DNA).
 13. The methodof claim 1 wherein the process is operated continuously.
 14. The methodof claim 1 wherein the molecules are extracted from the array ofobstacles.
 15. The method of claim 1 wherein the molecules are loadedusing electric fields.
 16. The method of claim 1 wherein the moleculesare extracted from the array of obstacles using electric fields.
 17. Themethod of claim 1 wherein the molecules are routed to the nextprocessing step after fractionation.
 18. A method of fractionatingcharged macro-molecules comprising: loading molecules into a matrix withan array of obstacles; applying to the matrix electric fields whoseamplitudes are constant in time; varying the field orientation with timeto create an asymmetrical electric field.
 19. The method of claim 18wherein the step of varying the field orientation with time to create anasymmetrical electric field comprises varying the field orientation withtime in such a manner that ∫[θ(t)]^(n+1)dt are not zero for every n,where θ(t) is field orientation with respect to the time-average fieldorientation, and n is any even integer larger than zero.
 20. The methodof claim 19 wherein the fields alternate between two fixed orientations.21. The method of claim 18 wherein the charged macro-molecules aredeoxyribonucleic acid (a.k.a. DNA).
 22. The method of claim 18 whereinthe process is operated continuously.
 23. The method of claim 18 whereinthe molecules are extracted from the array of obstacles.
 24. The methodof claim 18 wherein the molecules are loaded using electric fields. 25.The method of claim 18 wherein the molecules are extracted from thearray of obstacles using electric fields.
 26. The method of claim 18wherein the molecules are routed to the next processing step afterfractionation.
 27. An apparatus for fractionating chargedmacro-molecules comprising an array of obstacles and asymmetricallyalternating electric fields.
 28. The apparatus of claim 27 wherein theasymmetrically alternating electric fields comprise: an electric fieldwhich is alternating in direction as a function of time at a location inthe matrix, and which has a time average of its vector over many cycles,whereby the time integral of the electric field vector at the samelocation over the part of the cycles when it is instantaneously pointingto one side of the said time-averaged electric field vector is notspatially symmetric about the same said time-averaged vector with thetime integral of the electric field vector over the part of the cyclesat the same location when it is instantaneously pointing to the otherside of the same said time-averaged vector.
 29. The apparatus of claim27 wherein the asymmetrically alternating electric fields comprise:time-dependent electric fields {right arrow over (E)}(t) whose odd-orderintegrals over time, ∫|{right arrow over (E)}(t)|^(n){right arrow over(E)}(t)dt, are not at the time-average field orientation for every n,where n is any positive even integer.
 30. The apparatus of claim 27wherein the asymmetrically alternating electric fields comprise: firstand second electric pulses of first and second waveforms; the integralover time of one of the first or second pulses' amplitude larger thanthat of the other pulse; the orientation of the first electric pulsevarying between a first orientation and second orientation, and theorientation of the second electric pulse varying between a thirdorientation and forth orientation.
 31. The apparatus of claim 30 whereinthe first and second waveforms are square pulses.
 32. The apparatus ofclaim 31 wherein one of the square pulses is of higher amplitude thanthe other.
 33. The apparatus of claim 31 wherein one of the squarepulses is of longer duration than the other.
 34. The apparatus of claim27 wherein the asymmetrically alternating electric fields comprise:first and second alternating electric pulses of first and secondwaveforms; the integral over time of one of the first or second pulses'amplitudes larger than that of the other pulse; the first and secondelectric pulses applied at first and second fixed orientations.
 35. Theapparatus of claim 34 wherein the first and second waveforms are squarepulses.
 36. The apparatus of claim 35 wherein one of the square pulsesis of higher amplitude than the other.
 37. The apparatus of claim 35wherein one of the square pulses is of longer duration than the other.38. The apparatus of claim 27 wherein the asymmetrically alternatingelectric fields comprise: electric fields whose amplitudes are constantin time; the field orientation varying with time in such a manner that∫[θ(t)]^(n+1) dt are not zero for every n, where θ(t) is fieldorientation with respect to the time-average field orientation, and n isany even integer larger than zero.
 39. The apparatus of claim 38 whereinthe fields alternate between two fixed orientations.
 40. The apparatusof claim 27 wherein the charged molecules are deoxyribonucleic acid(a.k.a. DNA).
 41. The apparatus of claim 27 wherein the apparatus isoperated continuously.
 42. The apparatus of claim 27 wherein theapparatus comprises extraction structures for extracting fractionatedmolecules from the array of obstacles.
 43. The apparatus of claim 27wherein the apparatus comprises loading channel(s) for loadingmolecules.
 44. The apparatus of claim 27 wherein the molecules areextracted from the array of obstacles using electric fields.
 45. Theapparatus of claim 27 wherein the molecules are loaded into the array ofobstacles using electric fields.
 46. The apparatus of claim 27 whereinthe molecules are routed to the next processing step afterfractionation.